The long-term goal of this research is to determine the molecular mechanisms that regulate cell growth and differentiation. The molecular scaffold Kinase Suppressor of Ras 1 (KSR1) plays a critical role in regulating the intensity and duration of signaling through the Raf/MEK/ERKkinase cascade, directing its output toward either proliferative or adipogenic programs. A related family member, KSR2, may have both distinct and overlapping functions with KSR1. Deletion of KSR1 markedly reduces basal and AMP kinase (AMPK)- mediated glucose uptake in mouse embryo fibroblasts (MEFs). Expression of ectopic KSR1 restores uptake. In contrast to wild-type MEFs, the proliferative rate of KSR1'A MEFs is resistant to glucose deprivation. The inability of KSR"'" MEFs to respond appropriately to nutrient deprivation suggests the hypothesis that KSR proteins function as an organizing nodes for the coupling of cell nutritional status to mechanisms regulating cell proliferation and differentiation. Consistent with this hypothesis, KSR2"7' mice become obese and glucose intolerant. KSRTA mice, though lean, also demonstrate adipocyte hypertrophy and glucose intolerance. Coupled with the potent effect of KSR1 in regulating Raf and MEK activation, these observations suggest a previously unidentified, but physiologically important, interdependence between the Raf/MEK/ERK signaling cassette and mechanisms regulating cellular metabolism. The details of those mechanisms will be revealed through three specific aims: 1) Determine the relative ability of KSR1 and KSR2 to regulate ERK signaling and affect proliferation, differentiation, and metabolism. The biological function of each scaffold will be assessed in vitro by the expression of KSR1 and KSR2 mutants in KSR"'" MEFs. Tissue-specific glucose metabolism, and mitochondrial energy metabolism will all be assessed in KSR1"A and KSR2"'" mice. 2) Identify the mechanisms controlled by KSR1 and KSR2 to activate AMPK and control cell responses to energy stress. The subcellular distribution and activity of proteins regulating energy stress will be examined in cells in the presence and absence of KSR1 and KSR2. Knockout mice will be used to determine the in vivo effect of KSR1 and KSR2 on proteins regulating energy stress. 3) Identify the mechanisms activated in response to energy stress that affect cell proliferation. The effect of glucose deprivation on KSR1 and KSR2 phosphorylation will be evaluated. Proteins that negatively regulate KSR function in response to energy stress will be studied. The metabolism of mice lacking these negative regulators of KSR function will be evaluated. This research will provide insight into novel mechanisms regulating cell growth and metabolism. These studies may reveal important information about the factorscontributing to obesity and diabetes in humans.